The sequence-specific protein-DNA interaction between the eukaryotic transcription factor, ADR1, and its cognate DNS, UAS1, will be studied. The DNA-binding domain of ADR1 contains two Cys2-His2 zinc finger motifs that, along with a short N-terminal sequence, are necessary and sufficient for the high-affinity, specific protein-DNA interaction that is involved in regulation of the AHD2 gene in Saccharomyces cerevisiae. These two zinc fingers have each been studied in detail over the past granting period, using synthetic finger peptides and 1H homonuclear nuclear magnetic resonance (NMR) spectroscopy. Advances in multidimensional heteronuclear NMR spectroscopy make it possible to determine three-dimensional structure of large proteins and complexes than have been approachable using homonuclear spectroscopy. We propose to apply these techniques to the DNA-binding region has been over-expressed in E. coli. The expressed protein fragment retains high affinity for the UAS1 DNA sequence. The protein will be isotopically- enriched with 15N and/or 13C and heteronuclear NMR spectroscopy will be used to determine the structure of the protein both in the presence and absence of the UAS1 DNA sequence. In addition, the labelled material will allow us to examine dynamical properties of the protein, again in the presence and absence of DNA. Once a detailed description of the structure and properties of the wild- type protein-DNA complex has been obtained, mutants that have been shown to have altered DNA-binding properties will be examined. These studies will provide a wealth of information concerning the specific protein-DNA interactions of the ADR1 zinc fingers in particular and of the Cys2-His2 zinc finger motif in general. An ultimate goal of this research is the ability to design new zinc finger-containing proteins with new DNA binding specificities.